Uniparental genetic markers to investigate hybridization in wild-born marmosets with a mixed phenotype among Callithrix aurita and invasive species

The native marmoset of the Southeastern Atlantic Forest in Brazil is among the 25 most endangered primates of the world. Hybridization with alien species is one of its main threats registered since the early 2000s based on phenotype, so far, without genetic confirmation. Using uniparental molecular markers, we analyzed 18 putative hybrids, captured from 2004 to 2013 in different localities of the Atlantic Forest. A nine base pair deletion in the SRY gene of C. aurita was used to investigate paternal ancestry. Maternal ancestry was assessed by DNA sequencing of ca. 455 bp from the COX2 gene. Hybridization was confirmed for 16 out of the 18 marmosets since they inherited COX2 haplotypes of the alien C. penicillata or C. jacchus and the SRY deletion specific to C. aurita. Two individuals inherited both parental lineages of C. aurita, which is probably related to backcrossing or hybrid interbreeding. The direction of hybridization of females with the matrilineal lineage of invasive species with males descending from the native lineage was predominant in our sampling. This is the first time that hybridization between C. aurita and invasive species has been confirmed through genetic analysis.

www.nature.com/scientificreports/ Our main goal was to investigate the occurrence of in situ hybridization using uniparental markers amongst a sample of marmosets with a mixed phenotype, captured over nine years from different places of the Atlantic Forest and to assess whether there is a pattern in mating.

Results
Sample data. Eighteen marmosets considered putative C. aurita hybrids (Csp) were captured in three different localities in the state of Rio de Janeiro and one place in the state of São Paulo, Brazil, from 2004 to 2013 ( Fig. 1, Table 1).
Among the putative hybrids, there were 15 males and three females. Fifteen wild-born individuals of Callithrix aurita (six), C. jacchus (five) and C. penicillata (four) were sampled as positive controls (Table 1).
In general, the putative hybrids exhibited short ear tufts, in some cases fan-shaped with darker color. Commonly, the loss of the yellowish supra-cranial line peculiar to C. aurita was observed. The color of the body was lighter with eventual grooves (Fig. 2). These features confer to the individuals sampled a mixed pelage pattern of the tufts and the back, suggesting that they are hybrids between C. aurita and C. penicillata or C. jacchus 29,32,41 (since both alien species are observed at the sampling locations). SRY analysis. In our preliminary analysis to test the efficacy of the SRY primer set developed in this research to ascertain the patrilineal lineage of the putative hybrids, we sequenced the PCR products of one pure individual of each C. aurita (Ca), C. jacchus (Cj) and C. penicillata (Cp) used as a positive control and five putative hybrids of Callithrix sp. (Csp3, Csp4, Csp5, Csp6 and Csp7). We obtained the SRY nine base pair deletion 117_125delTAA GTA TCG exclusive to C. aurita in the Ca positive control and in the five Csp samples tested. At the same position is the insertion in the Cj and Cp positive controls, which presented identical DNA sequences (Fig. 3).
Afterward, using the same primer set labelled with 6-FAM fluorescent dye, an amplicon of the SRY of 196 bp was observed for all six Ca positive controls and 18 putative hybrids samples attesting to the patrilineal lineage of the native threatened species. For all nine positive controls of Cj and Cp an electropherogram peak of 205 bp was generated ( Table 1 provides an example of the electropherogram for each subset of obtained peaks. The image of the 33 peaks can be found in Supplementary Fig. S1 and S2 online).
The amplification of the SRY fragment in the eight female samples among the positive controls (Ca2, Cj3, Cj4, Cp1 and Cp3) and the putative hybrids (Csp1, Csp11, and Csp18) was possible due to the occurrence of placental anastomosis which allows hematopoietic chimerism resulting from the high incidence of twin gestation in callitrichids 42 .  www.nature.com/scientificreports/    www.nature.com/scientificreports/ 118,192.1 haplotype and did not cluster with any of the Callithrix sp. neither with our positive control samples. The second haplotype identified as HCp was shared by four of our positive controls, Cpe3_AY118196.1 from GenBank® and seven Callithrix sp. (Fig. 5). The third haplotype, HCp2, was shared by Cpe2_AY118195.1, and four of our Callithrix sp. samples (Fig. 5). For C. kuhli, one COX2 haplotype, AY118193.1, represented a specific branch (Fig. 5).
Each of the five haplotypes identified was separated by at least one variable of a total of 37 polymorphic sites. Haplotype diversity was 0.8205 ± 0,0356, and nucleotide diversity (π) was 0,0313 ± 0,0159. Among the haplotypes identified, there were 22 diagnostic variable sites for C. aurita, nine for C. penicillata and one for C. jacchus (see Supplementary Table S1  The unrooted phylogenetic tree, generated by the alignment of the COX2 sequences, including those from GenBank®, shows the greatest genetic variability of C. penicillata forming three well-defined clusters with posterior probabilities over 0.83 of node support (Fig. 5). One cluster is closer to that of C. geoffroyi (Cge_AY118119.1). Our Cp positive controls and seven Csp (12-18) clustered with Cpe3_AY118196. Four Csp (3-5 and 7) clustered with Cpe2_AY118.195.1, phylogenetically closer to C. jacchus. The sequences of none of the positive controls grouped with C. geoffroyi or C. kuhlii.

Discussion
Hybridization was undoubtedly confirmed for 16 out of the 18 free-living marmosets studied since all of them inherited the SRY deletion 117_125delTAA GTA TCG specific to C. aurita and COX2 haplotypes of C. penicillata or C. jacchus. The molecular markers of patrilineal and matrilineal ancestry combined here were suitable to attest hybridization in almost 90% of the sampled individuals. Two male individuals, identified as Csp6 and Csp8, from PARNASO in the Teresópolis municipality of Rio de Janeiro state, although exhibiting a mixed phenotype, inherited both parental lineages exclusive to C. aurita which is probably related to backcrossing or hybrid interbreeding. These two individuals were members of a group of C. aurita and C. penicillata, where the only adult male belonged to the native species 27 .
The hybrids sampled were distributed throughout the original area of occurrence of C. aurita, with one locality in São Paulo state at Biritiba Mirim and three localities of Rio de Janeiro state, in the municipalities of Teresópolis, Guapimirim and Petrópolis. At these localities, C. penicillata is present in almost 60% of the interspecific breeding, C. jacchus in 30% and less than10% of backcrossing to C. aurita was registered. Despite being a small sampling by location, the greater participation of C. penicillata in hybridization with C. aurita corroborates the expectation due to the similarities in the pattern of environmental fitness between both species 22 and also with the greatest ex situ reproductive success observed between these species when compared to C. jacchus 5 .
Although the fertility of the hybrids had already been confirmed experimentally through many directional matings through the six species of Callithrix 5 and in situ among C. penicillata and C. jacchus 24 , nothing is known about the fertility of free-living hybrids of C. aurita and still less about the patterns of in situ mating selection. The divergence of the Atlantic Forest marmosets occurred lightly more than 5 Ma in the Pliocene-Pleistocene scenario [43][44][45] . In birds, a typical young pair of sister species has only 1-2 Ma, and fertile hybrids still occur when species have nearly 7-17 Ma. Complete hybrid unfeasibility occurs when species are separated by approximately 11-55 Ma 46 . Therefore, considering only the phylogenetic proximity among the marmoset species, several kinds of crossings could result in viable and fertile hybrids representing an additional competitor to the threatened C. aurita.
We drew attention to the fact that most individuals inherited the nuclear DNA marker of C. aurita and that the mitochondrial DNA marker of one of the invaders cannot ensure that they represent the F1 generation without previous life history knowledge. The same limitation exists to confirm backcrossing or interbreeding among hybrids as observed from samples of Csp6 and Csp8 individuals who presented patrilineal and matrilineal DNA sequences of C. aurita but with phenotypes that correspond neither to the native species nor to the invasive ones (see Supplementary Fig. S5 online). Multiple nuclear markers are needed to attest to which generation they belong to and for a more complete understanding of the extent of introgression. Microsatellite markers 47-49 and a panel of SNPs developed from C. jacchus and C. penicillata 50 are already available and should be tested on C. aurita for this purpose.
The suspicion of hybridization based on the morphology of the Y chromosome and mixed phenotype in five samples 39 analyzed here (Csp3, Csp4, Csp5, Csp6 and Csp7) was now confirmed with the SRY and COX2 primer sets. Due to the subtle difference in the morphology among the Y chromosome of the native species and the invasive species and the polymorphism of this chromosome observed for the invasive species 35,38 , it is advisable to confirm through molecular genetics the inheritance of the C. aurita Y chromosome in hybrids based on cytogenetics 31 . Since SRY 117_125delTAA GTA TCG is specific to C. aurita, the SRY primer set is suitable for this purpose.
According to COX2, we found greater genetic variability in C. penicillata than in C. jacchus and C. aurita. Our C. penicillata samples, engendered three haplotypes forming well-defined clusters with above 83% node support. www.nature.com/scientificreports/ Considering that Cge_AY118119.1 (representing C. geoffroyi) and Cpe1_AY118194.1 (representing C. penicillata) are from GenBank® and that there is no other sample matching them, we will not discuss their proximity. However, the Cpe2_AY118195.1 and Cpe3_AY118196.1 haplotypes, which matched our samples and generated two distinct groups, showed greater distances between each other than the one shown by the Cpe2_AY118195.1 and the C. jacchus cluster. This unprecedented result may suggest hidden phylogenetic diversity that may be revealed through extensive phenotype-genetic research along the C. penicillata home range. Apparent directional mating was observed between males carrying the C. aurita Y chromosome with invasive females or at least female descendants of matrilineal lineage of the invaders. All 18 hybrids sampled over nine years in different places in the Atlantic Forest of RJ and SP carry the Y chromosome of C. aurita and 16, the COX2 haplotype of C. jacchus or C. penicillata. The occurrence of the small isolated populations in contact with the closely related invasive species in a fragmented habitat is much more likely to hybridize because of the difficulty of finding mates of the same species 17 , the same scenario experienced by C. aurita in the Atlantic forest. The population decline and the consequent reduction in the number of native females for breeding may lead to hybridization as an alternative strategy to carry on reproduction. Considering that in most primates, females are philopatric and males disperse, male-mediated asymmetric introgression is the most likely outcome 18 . The prevalence of males of one population/species mating with heterospecific females, instead of reciprocal crosses, has already been observed for other mammalian taxa. For example, the introduced American mink (Mustela vison) is more numerous, and males are larger than the European threatened native mink (Mustela lutreola) which favors them to mate with European mink females 51 . As male of C. aurita is larger than the invaders, it could be one factor favoring their success with congeneric females. Added to the reduction of encounters, habitat devastation is recognized for favoring hybridization, since the modification of the environment can hinder communication and recognition of conspecifics 52 . Hybridization in São Paulo and Rio de Janeiro states represented in our sample can be boosted by deforestation, which increased just this year by up to 400% in São Paulo and more than doubled in Rio de Janeiro 53 . The Atlantic Forest retains only 12.4% of its original vegetation 54 , which is highly fragmented, and the largest fragments generally do not exceed 50 hectares 53 . It is urgent to seek information in the field on populations of C. aurita that inhabit more extensive and preserved forest fragments to ascertain whether the establishment of exotic species occurs and what kind of interaction takes place between them. The pure populations of the native species can also act as a source of parental native genome that can mitigate hybridization effects 19 . In the municipality of Sapucaia, Rio de Janeiro, one of the few records of the occurrence of populations of C. aurita was made without the presence of aliens in the vicinity 55 .
This is the first genetic confirmation that hybridization takes place among C. aurita and the invasive species C. jacchus and C. penicillata over time, although based on sampling in a small extension of the species distribution. The uniparental molecular markers used, despite the limitations that we have already noticed, allowed basic aspects of hybridization to be raised based on newly generated data.
In the end, we highlight the fact that there are possibly among the hybrids studied here, individuals that carry approximately 50% and maybe more of the genomic composition of the threatened native species C. aurita. This percentage is among what is currently recommended by several researchers in this field as a reasonable limit for protection 56,57 representing an advance in relation to the minimum of 75% proposed in the intercross policy of 1996 57,58 . In Brazil, the recent creation of a captive breeding program 12 can provide the necessary support to deepen the genetic, reproductive and physiological research regarding C. aurita hybrids. However, to a coherent hybrid policy, ecological features and historical process that resulted in admixture individuals should be incorporated 57 . As already highlighted, we must bear in mind that each case of hybridization is peculiar and to be effective, conservation general rules must be reassessed 21,57 . Considering that the southeastern region of Brazil presents different scenarios where habitat loss and forest fragmentation coexist with some remaining but significantly large and continuous Atlantic Rain forested areas, we emphasize that relative to the hybridization among Callithrix aurita and invasive marmosets, we must think about it regionally.

Methods
Sampling location. Sampling of the pure individuals for positive controls occurred in areas of the Cerrado, Caatinga and of the southeastern region of the Atlantic Forest, the natural areas of occurrence of C. penicillata, C. jacchus and C. aurita, respectively. The positive controls were represented by six individuals of C. aurita, five C. jacchus and four C. penicillata, among which there were five females. All of them were wild born out of one C. jacchus (Cj1) born in captivity at Centro de Primatologia do Estado do Rio de Janeiro/CPRJ in 2009 (microchip number 030000007075), whose parents were wild born. Two individuals of C. jacchus were in captivity at the time of sample collection at the Wildlife Screening Center (Centro de Triagem de Animais Silvestres/CETAS/ IBAMA) in Recife city in the state of Pernambuco, Northeast Brazil (original habitat of this species) and one C. penicillata in CETAS/IBAMA of Brasília city in the Midwest region of Brazil (original habitat of this species). They are identified by the respective initials of the pure species (Ca, Cj and Cp) followed by an Arabic numeral ( Table 1).
The putative hybrid marmosets were sampled in areas of the Atlantic Forest, a highly threatened ecosystem, classified as a global hotspot of biodiversity, due to its exceptional concentration of endemic species and loss of more than 90% of its primary vegetation 59 and is the natural area of occurrence of C. aurita. The putative hybrids were identified with the acronym Csp (Callitrix sp.) followed by an Arabic numeral (Table 1).
Five males out of the eighteen putative hybrids studied here (Csp3, Csp4 and Csp5 from Guapimirim and Csp6 and Csp7 from PARNASO) have already been analyzed by cytogenetics and according to the morphology of their Y chromosome they were supposed to be hybrids fathered by a C. aurita male or a hybrid with the Y chromosome of this species 39  www.nature.com/scientificreports/ of the Callithrix species, it is difficult to attest patrilineal lineage solely based on cytogenetic data. For this reason, those samples were included for analysis using nuclear and mitochondrial markers. The free-living animals studied were captured in Tomahawk traps, baited with fruits, and sedated with ketamine hydrochloride (10 mg/kg) via intramuscular injection for morphological evaluation and blood sampling.
All research conducted followed guidelines for the ethical treatment of nonhuman primates approved by the Ethical Commission of the Instituto de Biologia Roberto Alcantara Gomes of Universidade do Estado do Rio de Janeiro (UERJ) for the Care and Use of Experimental Animals and adhered to Brazilian legislation number CEUA035/2014. Research permits were approved by the Brazilian Ministry of Environment, MMA/IBAMA/ SISBIO number 31570-5.
The study was carried out in compliance with the ARRIVE guidelines (https:// arriv eguid elines. org).
SRY and COX2 analysis. To investigate the paternal lineage we used the sex-determining region of the Y chromosome (SRY) gene based on GenBank® sequences (AF338377, AF338381 and AF338379) of Callithrix 40 to design consensus primers FSRY (5'-TAC AGG CCA TGC ACA GAG AG-3') and RSRY (5´-CTA GCG GGT GTT CCA TTG TT-3') for amplification through polymerase chain reaction (PCR), of an amplicon of ca. 200 bp, which includes the nine base pair deletion exclusive of C. aurita 40 . The organization of the mitogenome of the five Callithrix species was assumed to be free of NUMTS according to the high-quality base calls with no ambiguous nucleotides that could indicate a population of nuclear DNA of mitochondrial origin 50,60 .
To analyze the matrilineal lineage we used the primer set which flanks mitochondrial cytochrome c oxidase subunit II (COX2) gene following the advised PCR protocol 30 .
DNA was extracted from blood samples through phenol-chloroform protocol 61 . PCRs to amplify the SRY fragment contained 20-25 ng of DNA, 1-2 µM of each primer, 0.25-1 U of Taq DNA polymerase Platinum (Inv-itrogen®), 1X Taq buffer, 1.5 mM (for SRY)-2.5 mM (for COX2) MgCl 2 , 0.2 mM dNTPs and ultrapure water for a total reaction volume of 25 µL. The amplifications were carried out in a Veriti® 96-well Thermal cycler (Applied Biosystems, Co.) under the following conditions: a denaturation step of 94 °C for 5 min, followed by 30 cycles at 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min, and a final extension step of 72 °C for 10 min.
PCR products were checked by electrophoresis in 2% agarose gel dyed with ethidium bromide (10 mg/ml). The amplicons of SRY and COX2 were purified with Sephadex Resin and sequenced in an ABI PRISM 3500 (Applied Biosystem, Co).
Sequencing of the SRY amplicon was performed for three positive controls, one of each Callithrix species and five putative hybrids to check for the presence of the nine base pair insertions/deletions described in the pure species. Once it was confirmed, the forward primer was labelled with 6-FAM fluorescent dye, and all the PCR products were analysed in a ABI PRISM 3500 system (Applied Biosystem, Co) to identify the size of the amplicons using Geneious software. Our purpose was to make the analysis of putative hybrids more agile, eliminating the DNA sequencing step.
All samples were sequenced for the COX2 amplicon. The DNA sequences generated were aligned with Clustal W using the Megalign DNASTAR/Lasergene program, Inc. The GenBank® sequences of C. aurita (AY118189.1; AY118188.1), C. jacchus (AY118190.1; AY118191.1), C. penicillata (AY118194.1; AY118195.1; AY118196.1), C. geoffroyi (AY118192.1) and C. kuhlii (AY118193.1) were aligned with the haplotypes identified here to verify nucleotide and amino acid similarities using MEGA-X 62 . DNAsp6 63 was used to identify parsimony informative sites for each species and to estimate haplotype and nucleotide diversity. MRBAYES 3.2.1 64 was used to build the Bayesian tree to analyze phylogenetic relationships under an HKY85 substitution model using a Metropoliscoupled, Markov Chain Monte Carlo (MCMC). Four chains were run simultaneously for 500,000 generations in two independent runs, sampling trees every 100 generations. A burn-in of 1000 trees was carried out for each independent run. A consensus tree with nodal posterior probability support was obtained for five Callithrix species and Callithrix sp. samples.